Influence of embryonic stage on the transdifferentiation of chick neural retina cells in culture

Transcription

1 /. Embryo/, exp. Morph. Vol. 47, pp , 1978 \~J<) Printed in Great Britain Company of Biologists Limited 1978 Influence of embryonic stage on the transdifferentiation of chick neural retina cells in culture By D. I. DE POMERAI 1 AND R. M. CLAYTON From the Institute of Animal Genetics, University of Edinburgh, Scotland SUMMARY Neural retina cells from chick embryos up to 15 days of incubation can transdifferentiate in culture into both lentoids and pigment cells. Some transdifferentiation into pigment cells but none into lentoids was found in cultures of 17-day embryonic neural retina. No transdifferentiation occurred in cultures of neural retina from embryos immediately before hatching. In general, lentoids and pigment cells develop more rapidly and in greater numbers in cultures of neural retina from the earlier embryonic stages, and lens-specific crystallins also appear earlier and accumulate in greater amounts in these cultures. Delta crystallin accumulation is much greater in transdifferentiating cultures of early embryonic neural retina, whereas a and /? crystallins become proportionately more prominent in cultures of late embryonic neural retina. Traces of a and/? but not 8 crystallin are detectable in 60-day cultures of 17-day embryonic neural retina. Analogies between these results and the ontogeny of crystallin polypeptides in lens cells in vivo are discussed. INTRODUCTION Conversion of one specialized cell type into another occurs both in vivo and in vitro in the tissues of the vertebrate eye. The best-studied example of this in vivo is Wolffian lens regeneration from the dorsal iris epithelium in urodeles (reviewed by Yamada, 1977), though lens cells can also be derived from several other eye tissues in vivo (reviewed by Clayton, 1978 a). In early chick embryos the neural retina can regenerate from the underlying pigmented epithelium (Coulombre & Coulombre, 1965). During cell culture of eye tissues in vitro, lens cells may differentiate as rounded lentoid bodies composed of elongated, fibre-like cells wrapped around each other. Morphologically and ultrastructurally, lentoid cells resemble true lens fibres, and they also contain high concentrations of the lens-specific crystallins. Such lentoids may be derived from newly hatched chick lens epithelial (LE) cells in mass or clonal culture (Okada, Eguchi & Takeichi, 1971; Okada, Eguchi & Takeichi, 1973), from mass or clonal cultures of 8-day embryonic chick pigmented epithelium (PE) cells (Eguchi & Okada, 1973), 1 Author's present address: Department of Zoology, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.

2 180 D.I. DE POMERAI AND R. M. CLAYTON from mass cultures of neural retina (NR) cells from 8-day or 3 -day chick embryos (Okada, Itoh, Watanabe & Eguchi, 1975; Araki & Okada, 1977), and also from mass cultures of human foetal NR cells (Okada et al. 1977). In the case of LE cell cultures, lentoid development may be taken to reflect the normal process of fibre differentiation. In embryonic lens fibres, 8 crystallin predominates greatly over the a and /? crystallins; later, 8 crystallin declines in importance, and it is far less prominent in lens fibres laid down after hatching (Rabaey, 1962; Genis-Galvez, Maisel & Castro, 1968; Truman, Brown & Campbell, 1972). This in vivo trend is reflected under cell culture conditions in vitro, since embryonic LE cultures accumulate more 8 crystallin than do cultures of LE cells from newly hatched chicks (de Pomerai, Clayton & Pritchard, 1978). However, lens cells do not develop in vivo from embryonic chick neural retina or pigmented epithelium. The process by which these abnormal conversions can be effected under in vitro cell culture conditions has been termed transdifferentiation (Okada et al. 1975; Okada, 1976). Typically, prolonged culture periods are required (4-5 weeks for NR cultures, 6-10 weeks for PE cultures), during which time the cells will divide many times. Whether the transdifferentiation of NR cells might result from selection of a small sub-population of undifferentiated stem cells remains unclear, but in the case of PE cells it has been shown that clones derived from single pigmented cells can give rise to lentoid bodies (Eguchi & Okadi, 1973). Transdifferentiation of NR cells into pigmented epithelium may also occur, requiring some 3-4 weeks of culture (Itoh, Okada, Ide & Eguchi, 1975), and normally this process accompanies lentoid development in mass cultures of embryonic NR cells. One or both of these transdifferentiation pathways may be promoted by particular culture conditions in vitro, while other conditions suppress one or both pathways completely (Okada, 1976; Itoh, 1976; Clayton, de Pomerai & Pritchard, 1977; Clayton, \91%a,b). The production of lens-specific crystallins in the lentoids formed by transdifferentiation is of particular interest, and these proteins are well-characterized both biochemically and immunologically (reviewed by Clayton, 1974; Thomson et al. 1978#). We have previously shown that mature lentoid-containing cultures from 8-day embryonic NR do not contain the high levels of 8 crystallin characteristic of embryonic lens fibres, but rather show certain similarities to the cortical fibres of adult chickens in terms of their crystallin composition (de Pomerai, Pritchard and Clayton, 1977). Araki & Okada (1977) have recently reported that lentoid formation is much faster and 8 crystallin accumulation much greater in cultures from 3 -day embryonic chick NR, as compared with their 8-day counterparts. We now extend this type of analysis by comparing the patterns of crystallin accumulation during transdifferentiation of NR cells from a variety of embryonic stages. The results show a stage-related progression both in terms of the capacity to transdifferentiate, and in the crystallin composition of transdifferentiated lens fibres.

3 Transdifferentiation of chick neural retina 181 MATERIALS AND METHODS Fertile eggs were obtained from D. B. Marshall (Newbridge, Midlothian) and the Poultry Research Centre (Roslin, Midlothian). Sources of chemicals and apparatus are given below where appropriate. (1) Cell culture Neural retina was dissected, cleaned and dissociated with trypsin as described (de Pomerai et al. 1977; Okada et al. 1975). For 6-day embryos the entire dissection was performed under a dissecting binocular microscope; for 15-day and later embryos, great care was required to separate the neural retina from the closely adhering pigmented layer beneath it. The size of inoculum varied from 10 6 cells per dish in the case of 6-day embryonic NR up to about 10 8 cells per dish in the case of 17-day embryonic NR. These initial cell densities were chosen so that the cells which stuck down and survived were able to form large confluent patches within the first days of culture. Very few cells stuck down in the case of prehatching (21-day embryonic) NR, even with inocula as high as 10 9 cells per dish. In all cases the cells were established and grown in minimal essential medium containing Earle's salts, supplemented with 10% foetal calf serum, looi.u./ml penicillin, 100/tg/ml streptomycin, 2mM L-glutamine (all from GIBCO-Biocult Ltd., Paisley, Scotland) and 0-22% NaHCO 3. Cultures were maintained at 37 C in a humid atmosphere of 95% air:5% CO 2, the medium (4 ml per 6x1-5 cm tissue culture dish) being changed every 2-3 days. (2) Gel electrophoresis Cultures at various stages were washed several times in physiological saline and the cell sheet homogenized in a small volume of saline containing 20 HIM: /i-mercaptoethanol. The homogenate was centrifuged at g for 20 min to remove cell debris and the supernatant frozen in liquid N 2. Aliquots containing /tg protein were analysed on SDS-polyacrylamide gradient slab gels as described previously (Thomson et al ). Other aliquots were analysed by haemagglutination inhibition assays (see (4) below). (3) Antisera The antisera against purified a and 8 chick crystallin fractions were prepared as described previously (de Pomerai et al. 1977), and specific antisera against nine individual /? crystallin fractions were those characterized fully by Clayton & Truman (1974). For the purposes of the present work, an antiserum with broad anti-/? specificity was required, and this was obtained by pooling samples of all nine specific anti-/? sera. Antisera against a and 8 crystallin were further purified by means of columns containing respectively /? + # and pure 8 crystallins bound to CNBr-activated sepharose 4B (M. Jones & R. M. Clayton, in preparation). These antisera were both monospecific as judged by immunoelectrophoresis (de Pomerai et al. 1977).

4 182 D.I. DE POMERAI AND R. M. CLAYTON (4) Haemagglutination inhibition assays The haemagglutination inhibition assays used here are based on the method of Evans, Steel & Arthur (1974), as modified by de Pomerai et al. (1977). Originally we used sheep red blood cells coated with total day-old chick lens protein as indicator cells (de Pomerai et al. 1977), but we have since found that these do not always agglutinate well with anti-/? or anti-a sera, owing to the preponderance of 8 crystallin on the surfaces of the indicator cells. In the present work we have used indicator sheep red blood cells coated with adult chick cortex crystallins (where a and /? predominate over 8) when assaying for chick a and /? crystallins. Indicator cells coated with total day-old chick lens protein were used as previously when assaying for 8 crystallin. After incubation of twofold serial dilutions of the test sample (8 /tl) with 8 jtd antiserum (at such a dilution that it would just agglutinate the standard aliquot of indicator cells), the indicator cells (8 /d of a 2% suspension) were added and the mixture left to stand for several hours. The endpoint dilution at which the test sample ceased to inhibit agglutination was then noted and compared with that for identical assays on a known standard containing mg/ ml of total day-old chick lens protein. The concentration of a particular crystallin class (a, /? or 8, according to the antiserum used) in the test sample, relative to its concentration in the total day-old lens, is given by the equation: endpoint dilution for test sample x x 100 endpoint dilution for standard x protein concentration of test sample (mg/ml) From gel integration and other techniques, we estimate that total day-old chick lens crystallins comprise approximately 60 % 8 crystallin, 25 % /? crystallin and 15% a crystallin. Using these values as conversion factors, the concentration of each crystallin class in the test sample may be expressed as a percentage of the total soluble protein. The protein concentration in each test sample was estimated by means of scaled-down Lowry assays (Lowry, Rosebrough, Farr & Randall, 1951), using total day-old chick lens protein as a standard. All haemagglutination inhibition assays were performed at least in duplicate, and mean values from several sets of assays are plotted in Figs RESULTS (1) Development in culture of neural retina cells from various embryonic stages Figure 1 shows phase-contrast photographs of typical features during the development of 6-, 8-, 12-, 15-, 17- and 21-day embryonic chick NR cells in culture. In all but the last of these cultures, extensive confluent sheets of cells were formed within days. Very different inoculum sizes (see legend to Fig. 1) were required to achieve this, as the earlier embryonic cells stuck down

5 Transdifferentiation of chick neural retina 183 more readily and grew more vigorously than their later counterparts. In the case of 21-day embryonic (prehatching) NR, very few cells stuck down even at the highest densities attempted, and these grew very poorly. Groups of neuroblast-like cells were found overlying the cell sheet after about 8 days in the case of NR cultures from 6-, 8-, 12-, 15- and 17-day embryos; in some cases these groups were interconnected by long processes, which seemed especially welldeveloped in the case of 8-day material (see also Okada et al. 1975; Okada, 1976). By about 20 days, most cultures contained groups of small polygonal cells which have been designated 'prepigment cells' (Pritchard, de Pomerai & Clayton, 1978); these were especially marked in cultures from 6- and 17-day material but were absent in cultures from 21-day embryonic NR. By 30 days of culture both lentoids and patches of melanin-containing pigment cells were present in the 6-, 8-, 12- and 15-day embryonic NR cultures, but only pigment cells developed in cultures from 17-day material. Lentoids first appeared at 23, 25, 25 and 28 days respectively in the case of 6-, 8-, 12- and 15-day embryonic NR cultures. Differences in the frequency of lentoids and pigment patches became accentuated between 30 and 50 days of culture. Both occurred sparsely in NR cultures from 15-day embryos, more frequently in NR cultures from 12-day embryos, and abundantly in cultures from 8- and 6-day material. In 45-day cultures from 6-day embryonic NR, the larger lentoids were connected together by ridges of cellular material, the underlying cell sheet being almost entirely converted into pigmented cells (Fig. 2). No signs of transdifferentiation were seen in the cultures from 21-day embryonic NR, while in cultures from 17-day material, only transdifferentiation into pigment cells was observed. A very few structures which might represent small lentoids were observed at around 50 days in these latter cultures (Fig. 1; 17-day NR, days, B), but only traces of crystallin were detectable even 10 days later than this (see below). (2) Crystallin composition during lentoid development in cultures of neural retina cells from various embryonic stages The foregoing observations on the time of appearance and frequency of lentoids are reflected by the patterns of crystallin accumulation in these NR cultures (Fig. 3). The rate of crystallin accumulation in culture falls off with increasing developmental age of the NR starting material; thus some 60% of the total soluble protein is crystallin in the case of 52-day cultures from 6-day embryonic NR, as compared to 10% in the case of 15-day material after the same period in culture (Fig. 3). The proportions of the main crystallin classes (a, /? and 8) change as the total crystallin content rises; this is shown in Fig. 4 for each time-point used in Fig. 3. Generally speaking, 8 crystallin tends to increase in prominence during the culture period while a crystallin declines, with /? crystallin remaining approximately constant in the case of late embryonic NR cultures, or decreasing along with a in the case of early embryonic NR

6 184 D. I. DE POMERAI AND R. M. CLAYTON (a) Days in culture NR from 8-10 day day day (A) 6-day emb. 8-day emb. 12-day emb. 15-day emb. 17-day emb. 21-day emb. FIGURE 1 Development in culture of neural retina cells from different embryonic stages. NR cultures were established as described in Methods, using inocula of 10 6, 5 x 10 6,10 7, 5x 10 7, 10 s and 10 9 cells per dish for 6-, 8-, 12-, 15-, 17- and 21-day embryonic material, respectively. These cultures were examined by phase-contrast microscopy at 3-day intervals, and photographs of typical features are shown in Fig. 1 for each embryonic age at 8-10 days, days, days and days of culture. Two fields are shown for each of the last two time points, in order to illustrate the divergent modes of development towards pigment cells (column A) and lentoids (column B) respectively. Where no lentoids developed (17- and 21-day embryonic NR cultures), photographs of putative neuronal cells and processes have been substituted in column B.

7 Transdifferentiation of chick neural retina (b) Days in culture day(b) day (A) day (B) NRfrom 6-day emb. 8-day emb 12-day emb FIGURE \b cultures. The proportion of total crystallin represented by 8 is highest (up to 80 %) in cultures from 6-day embryonic NR, progressively lower in cultures from 8-day material (up to 60%) and 12-day material (up to 45 %), and lowest (only 30 %) in cultures from 15-day embryonic NR. The proportion of /? crystallin is small (5-15%) in cultures from 6-day material, varies from 20 to 40% in cultures from 8-day material, but remains relatively constant (25-30%) in

8 186 D.T. DE POMERAI AND R. M. CLAYTON Fig. 2. Lentoids and pigment cells in a 45-day culture of 6-day neural retina. A composite photograph of several adjacent fields showing lentoids and pigment cells in a 45-day culture from 6-day embryonic NR. Phase contrast microscopy and cell culture as for Fig. 1. cultures from later embryonic NR. The proportion of a crystallin is greatest (about 40 %) in cultures from 15-day embryonic NR, intermediate in the case of 12-day material (25-35 %) and 8-day material (20-30%), and lowest (10-15 %) in cultures from 6-day NR. The levels of a, /? and 8 crystallin are separately compared in Fig. 5 for cultures 6-, 8-, 12-, 15- and 17-day embryonic NR. The rates of accumulation of all three crystallin classes are greater in cultures of early than of late embryonic NR, but this trend is much more pronounced in the case of 8 crystallin. Thus at 52 days 8 crystallin reaches 20-fold higher levels in cultures from 6- as compared to 15-day material, whereas for a or /? crystallins this difference is only about twofold. The onset of detectable crystallin accumulation precedes the appearance of lentoids in the case of NR cultures from 6- and 8-day embryos (cf. de Pomerai et al. 1977), but in cultures from 12- and 15-day material, crystallins only reach detectable levels when the lentoids start to appear (Fig. 5). In cultures of 17-day embryonic NR, no crystallins were detectable after 38 days in culture, but traces ( % of the total soluble protein) of y^ and a but not 8 crystallins were found by 60 days (Fig. 5). Figure 6 shows the pattern of bands obtained by SDS-polyacrylamide gradient gel electrophoresis of soluble proteins extracted from NR cultures. In contrast to the gel system previously employed (de Pomerai et al. 1911), this method gives good resolution in the 8 crystallin region (M.W ; Piatigorsky, Zelenka & Simpson, 1974). Although a minor component comigrating with

9 Transdifferentiation of chick neural retina <>b 30- c A I! Days 3 B Days 3C Days 3 D Days A Days 4B Days 4C Days 4D Days Fig. 3. Quantitation of total crystallins in cultures of neural retina from different embryonic stages. At the indicated time points, cultures from 6-, 8-, 12- and 15-day embryos were washed and the soluble proteins extracted as described in Methods. Haemagglutination inhibition assays were used to quantitate the amounts of a, total ft and 8 crystallins in each extract. These amounts were expressed as percentages of total soluble protein (see Methods), and the total (a +/? T0T + #)% is shown as a bar for each time point in Fig. 3. Part A, levels of crystallin in cultures of 6-day embryonic NR; Part B, levels of crystallin in cultures of 8-day embryonic NR; Part C, levels of crystallin in cultures of 12-day embryonic NR; Part D, levels of crystallin in cultures of 15-day embryonic NR. Fig. 4. Proportions of a, ft and 8 crystallins in cultures of neural retina from different embryonic stages. The respective amounts of a, ft and 8 crystallins (see legend to Fig. 3) are here expressed as percentages of the total crystallin present at each stage in each culture series. For each bar, the total crystallin is taken as 100%, and the proportions of a, ft and 8 crystallins are shown by the extent of cross-hatching, of stippling, and of horizontal line shading, respectively. Each bar in Fig. 4 is placed immediately under the corresponding bar for Fig. 3. Part A, proportions of a, ft and 8 crystallins in cultures of 6-day embryonic NR; Part B, proportions of a, ft and 8 crystallins in cultures of 8-day. Part C, proportions of a, ft and 8 crystallins in cultures of 12-day embryonic NR; Part D, proportions of a, ft and 8 crystallins in cultures of 15-day embryonic NR.

10 188 D. I. DE POMERAI AND R. M. CLAYTON A - / / soliuble pi"otein ' / / f 1 //!, I / /// ^ zv l 0 JO Days in culture Fig. 5. Quantitation of a, ^ and #crystallins in cultures of neural retina from different embryonic stages. The levels of a, ft and tfcrystallins in NR cultures were determined by haemagglutination inhibition assays, and expressed as percentages of the total soluble protein at each time point. In addition to those points corresponding to the data in Figs. 3 and 4, additional points are given for the early stages of culture in each series, and also for 17-day embryonic NR cultures., 6-day embryonic NRcultures; # #, 8-day embryonic NR cultures; A""A, 12-day embryonic NR cultures; - -, 15-day embryonic NR cultures; T T, 17-day embryonic NR cultures. Part A, quantitation of 8 crystallin; Part B, quantitation of total (i crystallins; Part C, quantitation of total acrystallins. 8 crystallin is present even in very young cultures (e.g. 6-day cultures from 8-day embryonic NR), the later increase in prominance of this band (e.g. in 35- and 56-day cultures from 8-day embryonic NR) is probably due to the rise in genuine 8 crystallin (reaching 20% of the total soluble protein in 56-day cultures from 8-day NR; see Fig. 5). Beta crystallins are poorly resolved from neural retina components of similar molecular weight, but two bands comigrating with the a crystallins can be seen clearly in the later cultures from all embryonic stages. Figure 7 compares the protein compositions of 42-day cultures from 6-, 8- and 12-day embryonic NR. The relative amounts of a and 8 crystallins as shown by immunological methods (Fig. 5) are here clearly paralleled by the relative intensities of the putative cc and 8 crystallin bands.

11 Transdifferentiation of chick neural retina day emb. NR 8-day emb. NR r S f Delta Betas Alphas C=# 1 wm T* JC 12-day emb. NR day emb. NR Fig. 6. Protein composition of cultures of neural retina from different embryonic stages. Soluble proteins were extracted from NR cultures at various stages and samples ( fig) were run on SDS polyacrylamide gradient slab gels as described in Methods. On each slab gel, two samples of total day-old lens protein were also run as a standard, and the crystallin bands were identified according to Thomson et al. (1978 a, b). Since the gels photographed in Fig. 6 came from a number of different runs, the positions of the main a and 8 bands are marked by arrows for each slab, and only in one case is the day-old protein standard (S) also shown. Typical protein profiles are shown for 6-, 8-, 12- and 15-day embryonic NR cultures over a range of time points from 6 to 56 days of culture; for each sample, the number of days in culture is given in arabic numerals above the corresponding gel slot. Slots from the same gel are grouped together. EMB 47

12 190 D.I. DE POMERAI AND R. M. CLAYTON S -Delta Betas Alphas Fig. 7. Protein composition of 42-day cultures of 6-, 8- and 12-day embryonic NR. 80 fig samples of soluble protein from 42-day cultures of 6-, 8- and 12-day embryonic NRwere run on an SDS polyacrylamide gradient slab gel and stained as described in Methods. Total day-old chick lens proteins (40/<g) were also run on the same slab, and the resulting crystallin profile (S) is shown alongside. Arrows indicate the positions of a and 8 crystallin bands, and the j3 crystallin region is also shown. Arabic numerals here indicate the number of days of embryonic development for the starting NR, all cultures being incubated for the same period (42 days) in vitro. DISCUSSION The transdifferentiation of embryonic chick NR cells in culture into pigmented epithelium and lens fibres (lentoids) shows several progressive changes according to the developmental age of the starting material. The range of developmental ages tested in the present work (6- to 21-day embryonic NR) may be extended back to earliei stages by comparing our results with the data of Araki & Okada (1977) on transdifferentiation in cultures from 3 -day embryonic chick NR. Several differences between cultures from 3 -day NR and from 8-day NR were pointed out by Araki & Okada (1977), and our present data indicate that these represent consistent trends. In particular, early embryonic neural retina cells grow more vigorously and require less time for transdifferentiation into both lens and pigment cells, than do their later embryonic counterparts (see Results). Moreover, in cultures from early embryonic NR, almost all the cells transdifferentiate either into lentoids or pigmented epithelium (for 3 -day material, see Araki & Okada, 1977; for 6-day material, see Fig. 2), whereas transdifferentiation events of either type are much rarer in cultures from later (e.g. 15-day) embryonic NR. The case of 17-day embryonic NR is particularly interesting; here, transdifferentiation into lens cells is almost completely suppressed (no convincing lentoids and very low levels of crystallin; Figs. 1 and 5),

13 Transdifferentiation of chick neural retina 191 but transdifferentiation into pigment cells is still possible and may even be slightly enhanced in comparison with cultures of 15-day NR (Fig. 1). Neither type of transdifferentiation was observed in cultures from prehatching (21-day embryonic) NR cells (Fig. 1), nor in cultures from 1-, 2- and 7-week adult chicken NR cells (de Pomerai, unpublished observations). Several consistent trends are also observable in the crystallin composition of lens cells formed by transdifferentiation in cultures of NR from different developmental stages. The accumulation of crystallin is much faster and starts much sooner in the case of NR cultures from early embryos (for 3 -day material, see Araki & Okada (1977); for 6-day and later stages, see Results and Fig. 3). The content of 8 crystallin is also much higher in cultures of early embryonic NR. The data of Araki & Okada (1977) indicate that 8 predominates over a crystallin by approximately 20 to 1 in 30-day cultures from 3 -day material. In 45- and 52-day cultures from 6-day embryonic NR the ratio of 8:CL crystallin is about 7:1 (Fig. 4), and this 8:a ratio further declines to about 3:1, 2:1 and 1:1 respectively in 50- to 56-day cultures from 8-, 12- and 15-day embryonic NR (Fig. 4). Araki & Okada (1977) gave no data on the /? crystallin content of cultures from 3 -day embryonic NR, but one might expect the value to be rather low from the trend seen in Fig. 4, where /? comprises only 5-8 % of the total crystallin in cultures from 6-day embryonic NR after days in culture. This predominance of 8 crystallin in cultures of early embryonic NR, and the increasing contribution of a and {} crystallins in cultures from later embryonic NR (Fig. 4), are paralleled by a similar trend in cultures of lens epithelial (LE) cells from various stages in chick development. Here, 8 crystallin accumulation was greatest in cultures of 12-day embryonic LE cells, intermediate in cultures of 15-day embryonic LE cells, and poorest in cultures of newly hatched LE cells (de Pomerai et al. 1978). A correlation between the growth rate and crystallin composition of these LE cultures was indicated, and experiments were suggested which could test for any causal link between these two parameters (Clayton, 1978b; de Pomerai et al. 1978). Differential growth rates might also influence the crystallin composition of lens cells formed in transdifferentiating NR cultures. It should be noted that LE and NR cultures set up from the same stage produce quite different proportions of the main crystallin classes (e.g. high 8 in 12- or 15-day embryonic LE cultures, but low 8 in 12- or 15-day embryonic NR cultures). Although the amounts of 8 crystallin produced in LE cultures from different stages may reflect the normal differentiation into fibres which those LE cells would undergo in vivo (de Pomerai et al. 1978), this is clearly not the case for NR cultures. A rigorous comparison of growth curves for cultures of both LE and NR cells from different developmental stages would help to establish the correlation between growth rate and crystallin composition, but in itself such a study could neither prove nor disprove the hypothetical link between them. One possible source of evidence on this score would be an 13-2

14 192 D.I. DE POMERAI AND R. M. CLAYTON investigation of the effect on crystallin composition of a variety of culture conditions (Okada, 1976; Itoh, 1976; Clayton et al. 1977) which affect the growth rate of both types of culture. Preferably, both growth-promoting and growth-inhibiting conditions should be tested to see whether these lead to an increase and a decrease respectively in the proportion of crystallin synthesized. The authors would like to thank D. B. Marshall Ltd. and the Poultry Research Centre for supplying fertile eggs; Drs I. Thomson, D. J. Pritchard, P. G. Odeigah, J. F. Jackson, D. E. S. Truman and J. C. Campbell for useful discussions and suggestions during the course of this work; and the M.R.C. and C.R.C. for grants in support of this research. D. de P. is in receipt of an M.R.C. postdoctoral training fellowship. REFERENCES ARAKI, M. & OKADA, T. S. (1977). Differentiation of lens and pigment cells in cultures of neural retinal cells of early chick embryos. Devi Biol. 60, CLAYTON, R. M. (1974). Comparative aspects of lens proteins. In The Eye (ed. H. Davson & L. T. Graham, Jnr.), vol. 5, pp New York and London: Academic Press. CLAYTON, R. M. (1978O). Divergence and convergence in lens cell differentiation: regulation of the formation and specific content of lens fibre cells. In Stem Cells and Tissue Homeostasis (ed. B. I. Lord, C. S. Potten & R. J. Cole). Cambridge University Press. (In the Press.) CLAYTON, R. M. (19786). Genetic regulation in the vertebrate lens cell. In Mechanisms of Cell Change (ed. J. Ebert & T. S. Okada). Wiley. (In the Press.) CLAYTON, R. M. & TRUMAN, D. E. S. (1974). The antigenic structure of chick /? crystallin subunits. Expl Eye Res. 18, CLAYTON, R. M., DE POMERAI, D. I. & PRITCHARD, D. J. (1977). Experimental manipulation of alternative pathways of differentiation in cultures of embryonic chick neural retina. Devi, Growth and Differ. 19, COULOMBRE, J. L. & COULOMBRE, A. J. (1965). Regeneration of neural retina from the pigmented epithelium in the chick embryo. Devi Biol. 12, EGUCHI, G. & OKADA, T. S. (1973). Differentiation of lens tissue from the progeny of chick retinal cells in vitro: A demonstration of a switch of cell types in clonal cell culture. Proc. natn. Acad. Sci. U.S.A. 70, EVANS, J., STEEL, M. & ARTHUR, E. (1974). A haemagglutination inhibition technique for detection of immunoglobulins in supernatants of human lymphoblastoid cell lines. Cell 3, GENIS-GALVEZ, J. M., MAISEL, H. & CASTRO, J. (1968). Changes in chick lens proteins with ageing. Expl Eye Res. 7, ITOH, Y. (1976). Enhancement of differentiation of lens and pigment cells by ascorbic acid in cultures of neural retinal cells of chick embryos. Devi Biol. 54, ITOH, Y., OKADA, T. S., IDE, H. & EGUCHI, G. (1975). The differentiation of pigment cells in cultures of chick embryonic neural retinae. Devi, Growth and Differ. 17, LOWRY, O. H., ROSEBROUGH, N., FARR, A. & RANDALL, R. (1951). Protein measurements with the Folin-phenol reagent. /. biol. Chem. 193, OKADA, T. S. (1976). Transdifferentiation of cells of specialised eye tissues in cell culture. In Tests of Teratogenicity in vitro (ed. J. Ebert & M. Marois), pp Amsterdam: North Holland Press. OKADA, T. S., EGUCHI, G. &TAKEICHI, M. (1971). The expression of differentiation by chicken lens epithelium in in vitro cell culture. Devi Growth and Differ. 13, OKADA, T. S., EGUCHI, G. & TAKEICHI, M. (1973). The retention of differentiated properties of lens epithelial cells in clonal cell culture. Devi Biol. 34, OKADA, T. S., ITOH, Y., WATANABE, K. & EGUCHI, G. (1975). Differentiation of lens in cultures of neural retinal cells of chick embryos. Devi Biol. 45,

15 Transdifferentiation of chick neural retina 193 OKADA, T. S., YASUDA, K., HAYASHI, M., HAMADA, Y. & EGUCHI, G. (1977). Lens differentiation in cultures of neural retinal cells of human fetuses. Devi Biol. 60, PIATIGORSKY, J., ZELENKA, P. & SIMPSON, R. T. (1974). Molecular weight and subunit structure of delta-crystallin from embryonic chick lens fibers. Expl Eye Res. 18, DE POMERAI, D. I., CLAYTON, R. M. & PRITCHARD, D. J. (1978). Delta crystallin accumulation in chick lens epithelial cultures: dependance on age and genotype. Expl Eye Res. (In the Press.) DE POMERAI, D. I., PRITCHARD, D. J. & CLAYTON, R. M. (1977). Biochemical and immunological studies of lentoid formation in cultures of embryonic chick neural retina and dayold chick lens epithelium. Devi Biol. 60, PRITCHARD, D. J., DE POMERAI, D.I. & CLAYTON, R. M. (1978). 'Transdifferentiation' of chicken neural retina into lens and pigment epithelium in culture: controlling influences. /. Embryol. exp. Morph. (In the Press.) RABAEY, M. (1962). Electrophoretic and immunoelectrophoretic studies on the soluble proteins in the developing lens of birds. Expl Eye Res. 1, THOMSON, I., WILKINSON, C. E., BURNS, A. T. H., TRUMAN, D. E. S. & CLAYTON, R. M. (1978o). Characterisation of chick lens soluble proteins and the control of their synthesis. Expl Eye Res. 26, THOMSON, I., WILKINSON, C. E., JACKSON, J. F., DE POMERAI, D. I., CLAYTON, R. M., TRUMAN, D. E. S. & WILLIAMSON, R. (19786). Characterisation of chick lens crystallin mrna and its cell-free translation during normal development and transdifferentiation of neural retina. Devi Biol. (In the Press.) TRUMAN, D. E. S., BROWN, A. G. & CAMPBELL, J. C. (1972). The relationship between ontogeny of antigens and the polypeptide chains of the crystallins during chick lens development. Expl Eye Res. 13, YAMADA, T. (1977). Control Mechanisms in Cell-type Conversions in Newt Lens Regeneration. Monographs in Developmental Biology (ed. A. Wolsky) 13, Basel: S. Karger. (Received 8 March 1978) 13-3

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